Purification, amino-terminal sequence and functional properties of a 64 kDa cytosolic protein from heart muscle capable of modulating calcium transport across the sarcoplasmic reticulumin vitro

Abstract

In previous studies we have described the inhibitory action of a cytosolic protein fraction from heart muscle on ATP-dependent Ca2+ uptake by the sarcoplasmic reticulum (SR); further this inhibition was shown to be blocked by an inhibitor antagonist, also derived from the cytosol (Narayanan et al., Biochim. Biophys. Acta. 735: 53-66, 1983; Can. J. Physiol. Pharmacol. 67: 999-1006, 1989). Here we report the complete purification of the antagonist protein (AP) and characterization of its functional properties. AP was purified to homogeneity from rabbit heart cytosol using two procedures, one utilizing sequential DE52-cellulose and hydroxylapatite chromatography, and the other utilizing anion exchange chromatography on Mono Q HR 5/5 column in a Pharmacia FPLC system. The purified AP has an apparent molecular weight of 64 kDa; it is made up of about 43% hydrophobic and 57% hydrophilic residues with the following amino-terminal sequence: E-A-H-K-S-E-I-A-H-R-F-N-D-V-G-E-E-H-F-I-G-L-V-L-I-T-F-S-Q-Y-L-Q-K-X-P-Y- E-E-H-A . This partial amino acid sequence data indicate strong sequence homology to serum albumin (sequence homology: 85% to rat serum albumin and 74% to sheep and bovine serum albumin). The purified AP caused concentration-dependent-blockade of the inhibition of Ca2+ uptake by SR observed in the presence of the cytosolic Ca2+ uptake inhibitor protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Appearance of Cytosolic Albumin-Like Protein in Microsomal Fraction of Isolated Rabbit Heart during Global Ischemia.

“…We electrophoretically isolated in the microsomal fraction a 65-kDa protein that showed an apparent increase in response to ischemia and a decrease after reperfusion. The amino acid sequence in the N-terminal region was found to resemble the antagonist protein described by Xu and Narayanan [7]. The antagonist protein reported by Xu et al was present in the soluble cytosolic fraction, whereas the 65-kDa protein described in this report was isolated from the microsomal fraction of cardiac membranes.…”

“…The antagonist protein reported by Xu et al was present in the soluble cytosolic fraction, whereas the 65-kDa protein described in this report was isolated from the microsomal fraction of cardiac membranes. As suggested by Xu and Narayanan for muscle albumin [7], this 65-kDa cytosolic albumin may associate with cardiac membranes during ischemia.…”

“…which resembled the antagonist protein (cytosolic Ca2+ uptake inhibitor antagonist protein) found in rabbit heart cytosol (Xu and Narayanan (1994) Mol. Cell.…”

“…Cell. Biochem ., 132, [7][8][9][10][11][12][13][14]. Thus, Ischemia may induce cytosolic albumin to associate with the microsomal fraction of cardiac membranes.…”

“…They identified this antagonist protein as j3-actinin, now called muscle albumin. Xu and Narayanan [7] recently determined the amino acid sequence in the N-terminal region of this antagonist protein to be E-A-…”